**4. Results**

90 Recent Advances in Crystallography

**3. Materials and methods** 

**Figure 3.** Experimental results: (*a*) section of a 4x6 X-CHIP with a two-dimensional optimization of two crystallization conditions for native PA0269, taken two weeks after initial set up (*b*) crystals of EphA3 grown overnight, crystal size approximately 250μm in length (*c*) On-the-chip diffraction image for an EphA3 crystal, collected on a Rigaku FR-E rotating anode with R-AXIS HTC detector (*d*) Part of an experimental electron density map generated using the SAD PA0269SM data set collected directly from the crystal grown on the X-CHIP, superimposed with the protein Cα-trace, shown as a black solid line.

Previously investigated targets, the protein kinase domain of human Ephrin Receptor Tyrosine Kinase A3 (EphA3) (Davis *et al.*, 2008) and the *Pseudomonas aeruginosa*  alkylhydroperoxidase D protein (PA0269) (McGrath *et al.,* 2007) were selected as model proteins to demonstrate the feasibility of crystal growth and *in situ* data collection using the X-CHIP. Following several rounds of on-chip optimization, both projects were Two important aspects of the described system were investigated throughout this study; the capacity of the chip to produce diffraction quality crystals and the feasibility of diffraction data acquisition (*in situ*) of sufficient quality for *de novo* structure determination. To assess the first task, the reproducibility of previous crystal hits obtained by sitting drop vapor diffusion technique was tested. For both EphA3 and PA0269 projects, vapor diffusion crystallization conditions resulted in high-quality crystals on the X-CHIP (Fig. 3*a*, 3*b*). For PA0269, on-chip optimizations further improved the crystal size and quality and decreased the number of crystals per drop (Fig. 3*a*). These results demonstrated that the X-CHIP can be successfully used to obtain and optimize crystallization hits and grow single crystals that are large enough for straightforward data collection.

Proof-of-concept experiments for on-the-chip data collection were carried out on the rotating anode source and the synchrotron beamline. The initial data collection trials on the in-house X-ray source led to the acquisition of a complete EphA3 data set. While the experiment was conducted at room temperature, diffraction data could still be obtained with sufficient completeness, even for crystals of such low symmetry space group as P21 (Table 1). At the synchrotron beamline, data sets for EphA3, PA0269 and a PA0269 selenomethionine derivative (SAD) were collected. The high sensitivity and ultrafast readout time of the *Pilatus 6M* detector allowed complete data sets to be collected quickly at room temperature without significant degradation of the sample and with excellent processing statistics. Owing to the finely focused beam (50 x 50μm), it was possible to collect

#### 92 Recent Advances in Crystallography

data from multiple small crystals grown within the same drop, without any obvious impact on the diffraction quality of neighboring crystals. A particularly interesting result can be observed by comparing the mosaic spread between the X-CHIP and the benchmark data (*i.e.* cryo loop) in Table 1. It is evident that the mosaic spread was consistently lower for data set experiments collected using the chip, and in the case of PA0268 was as low as 0.046o. Furthermore, based on resolution range alone, EphA3 crystals only started showing radiation damage after as much as ten minutes of continuous X-ray exposure, more than twice the time needed for obtaining a full data set (data not shown).


Benchmark data is in *italic font.*

Values in parentheses refer to the highest resolution shell.

\*Single-wavelength anomalous dispersion (SAD) data collection, using anomalous signal from seleno-methionine. † Completeness of 99% was achievable from the same set of crystals with a total oscillation angle of 140 degrees.

**Table 1.** Summary of selected data sets.

For crystallization of EphA3 and PA0269, paraffin oil was used to coat the crystallization drops after protein and precipitant solution had been dispensed. Other oils have been explored, such as Hampton's Al's Oil (50/50 paraffin/silicon oil mixture), silicon oil and a 50/50 mix of paratone/paraffin oils. Higher viscosity oils (paraffin, paratone/paraffin) performed better on the X-CHIP by being highly restricted to the hydrophobic ring boundaries. The thinner silicon oil was found to flow outside of these boundaries causing drop merging. Al's oil required more careful application compared to higher viscosity oils, but proved to stay within the hydrophobic boundaries. Crystallization conditions containing ethanol, 2-methyl-2,4-pentanediol (MPD) and detergents were also tested on the X-CHIP. Ethanol tolerance was tested with a 5-30% gradient using paraffin oil as a cover. The phase separation within the crystallization drops remained intact for the entire gradient range. Crystallization drops containing MPD in combination with different oils tolerated up to 8% before they began to disperse beyond the hydrophobic area the hydrophobic area. While this can exclude some MPD based conditions from being used on the X-CHIP, the impact on the overall versatility is low since most commercially available initial screens from Hampton Research and Emerald Biosystems (e.g. Wizard I&II, Index, Crystal) only have an average of 5-6% total conditions containing MPD. Detergent tolerance was tested with n-dodecyl-N,N-dimethylamine-N-oxide (DDAO) and n-octyl-β-D-glucoside in combination with a paraffin oil covering. Separation between the phases remained intact with the 0.05% n-octyl-b-D-glucoside condition but, DDAO was not tolerated even at very low concentrations.

#### **5. Discussion**

92 Recent Advances in Crystallography

Crystal mount

High resolution

Data Collection

Benchmark data is in *italic font.*

shell (Å)

X-ray Source Rigaku

Detector R-AXIS

Sample

data from multiple small crystals grown within the same drop, without any obvious impact on the diffraction quality of neighboring crystals. A particularly interesting result can be observed by comparing the mosaic spread between the X-CHIP and the benchmark data (*i.e.* cryo loop) in Table 1. It is evident that the mosaic spread was consistently lower for data set experiments collected using the chip, and in the case of PA0268 was as low as 0.046o. Furthermore, based on resolution range alone, EphA3 crystals only started showing radiation damage after as much as ten minutes of continuous X-ray exposure, more than

**EphA3** *EphA3* **EphA3** *PA0269*

Temperature (K) 295 *100* 295 *100* 295 295

λ (Å) 1.54 *1.00* 1.00 *0.97934* 1.00 0.97938

Space group P21 *P21* P21 *P6322* P6322 P6322 Resolution (Å) 2.00 *1.93* 1.95 *1.75* 1.95 1.95

Time (min) 100 *29* 5.3 *74* 3.0 3.3 ∆φtotal (o) 100**†** *190* 160 *185* 90 100 Mosaic spread (o) 0.100 *1.048* 0.360 *0.364* 0.046 0.160 Completeness (%) 85.3**†** *98.4* 96.2 *99.8* 100 100 Multiplicity 2.4 *3.6* 2.7 *10.6* 9.5 10.3

\*Single-wavelength anomalous dispersion (SAD) data collection, using anomalous signal from seleno-methionine. † Completeness of 99% was achievable from the same set of crystals with a total oscillation angle of 140 degrees.

ID-17 APS

Pilatus 6M

(2.05- 1.95)

(34.6)

*ID-17 APS* 

*ADSC Q210* 

*(1.84- 1.75)* 

*(3.5)* 

*7.5 (49.9)* 

*BM-17 APS* 

*MarCCD M300* 

> *(2.03- 1.93)*

*SM\** **PA0269 PA0269S**

*Loop* X-CHIP X-CHIP

ID-17 APS

Pilatus 6M

(2.05- 1.95)

12.1

8.2

(2.7) 20.8 (3.9)

(47.5) 6.9 (50.2)

**M \*** 

ID-17 APS

Pilatus 6M

(2.05- 1.95)

twice the time needed for obtaining a full data set (data not shown).

method X-CHIP *Cryo Loop* X-CHIP *Cryo* 

FR-E

HTC

(2.10- 2.00)

<I/σ(I)> 4.5 (2.2) *12.9 (5.0)* 7.5 (2.7) *19.4* 

Rmerge (%) 8.8 (34.6) *4.9 (9.7)* 10.9

Values in parentheses refer to the highest resolution shell.

**Table 1.** Summary of selected data sets.

The series of initial experiments on the X-CHIP crystallization platform described above demonstrated the chip's applicability for high-throughput protein crystallography and provided insight into the benefits and limitations of this system. Crystallization using the microbatch method on the chip was shown to be suitable for crystal growth and also offered additional benefits. Oil covered drops evaporate very slowly (days to weeks), simplifying both manual and automated set up. Furthermore, changing the composition of the top oil layer with various oil mixtures makes it possible to vary the rate of water evaporation over a wide range, adding another favorable dimension to crystallization screening (D'Arcy *et al.*, 2004). Inherently, the system is economical since crystallization hit determination and optimization trials require up to five times less volume of protein sample and five hundred times less reagent solution than standard vapor diffusion methods. Theoretically, the volumes can be decreased even further by incorporating robotic liquid handling systems, but are currently limited by the accuracy of manual dispensing. In addition, the simplicity of the device results in low manufacturing costs and the platform design eliminates the time and expenses associated with cryogenic techniques. The small size of the chip offers more convenient and faster visual inspection, as all the crystallization drops can be viewed under a microscope simultaneously. Furthermore, the system design provides a non-invasive means of diffraction testing and screening, as the developed device can be mounted on most in-house and synchrotron beamline data acquisition systems without any modification of

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the chip or adjustments to that system. These capabilities of the X-CHIP make it a potentially useful platform for high-throughput initiatives such as fragment-based screening by co-crystallization.

The X-CHIP system has the potential to completely remove the "user factor" between crystal growth and X-ray diffraction data collection, eliminating crystal manipulation. The feasibility of *in situ* data collection has significant implications. Firstly, data collection at room temperature eliminates the need for the tedious and often limiting step of cryocondition optimization with the added advantage that crystal structures determined at room temperature are more representative of the physiological state. Additionally, experimentally obtained SAD data displayed excellent processing statistics, clearly of sufficient quality for *de novo* structure determination. Interestingly, in at least one of the cases investigated, undisturbed crystals have shown significantly lower mosaic spread than that of cryogenically frozen samples, suggesting the potential application of this system to samples of high sensitive or ones with a large unit cell (Table 1). Once mounted on the goniometer, navigation along the chip and alignment of any crystal in the drops is quite straightforward, presenting the potential for data collection in a high-throughput mode. This approach eliminates the necessity for mounting of individual loops as in conventional robotics systems and may save hours of valuable synchrotron beam time. Finally, the elimination of manual crystal handling opens the opportunity for full automation of the crystallization to data acquisition pipeline allowing streamlining of the entire process.

Current developments on the project are aimed at scaling down the drop volumes of the X-CHIP system. Attempting to do so using manual set up has proven to be challenging, but application of a liquid handling robotics system can address this issue. The *Mosquito*  crystallization robotic system (Molecular Dimensions Ltd., Suffolk, UK) has already been used to successfully set up crystallization experiments with total drop volumes as low as 200nl. The X-CHIP is also being applied to the crystallization trials of additional protein targets. As a point of interest, experiments with highly sensitive and/or small crystal samples could greatly benefit from the use of this system, as the non-invasive data collection approach would likely resolve many problems that arise from crystal handling. We are also exploring the application of the chip for projects in which low mosaic spread is essential for successful outcome.
